Abstract

With growing environmental concerns, industries are increasingly relying on the use of biofuels. Biodiesel storage tanks are susceptible to water infiltration that often results in the formation of biofilms containing bacteria and fungi. Biofilms may clog pipes, degrade fuel, or corrode storage tanks. We set out to engineer a “seek, aim, and destroy” system for the remediation of microbial biofilm. Pseudomonas aeruginosa, commonly found in fuel biofilms, releases the quorum sensing molecule N-butanoyl-L-homoserine lactone (C4-HSL). Our engineered E. coli cells express CheZ protein in response to a concentration gradient of C4-HSL to activate the flagella motors and propel the cells towards the biofilm. In addition, the engineered E. coli expresses chitinase on its surface and secretes cinnamaldehyde. Chitinase breaks down chitin in the fungal cell walls, increasing the ability of cinnamaldehyde to destroy the fungi. Cinnamaldehyde also eliminates bacteria, thus remediating the biofilm.

Our Approach

We split our project up into three parts: Detect, Deliver, and Destroy

Detect C4-HSL, using the RhlR receptor gene.

Deliver the plasmid to the biofilm using E. coli's natural chemotactic ability by expressing the protein CheZ

Destroy the bacterial and fungal biofilm by deploying two chitinase constructs to destroy chitin in the fungal cell walls, then utilizing cinnamaldehyde to kill the bacteria and fungi, which prevents biofilm formation.

The Problem

There are three clear distinct layers that form in fuel containment systems: A water layer, the sludge layer, and the actual fuel layer. The formation of these layers usually takes a few weeks, but over time, water seeps into these underground tanks due to imperfections with storage techniques. With this water, microorganism contamination also comes into the mix. Now, a perfect environment for these microorganisms is present, because the water provides the perfect living conditions, and the biofuel contains high amounts of FAMEs (Fatty Acid Methyl Esters) for the microorganisms to feed on. Due to the density difference between fuel and water, the fuel sits on top, the water sits on the bottom, and the microorganisms sit in the middle forming the sludge, which is more commonly known as a biofilm.

Detect and Deliver

Our microbe will tumble through the fuel searching for C4-HSL, a molecule secreted by Pseudomonas aeruginosa, a bacteria commonly found in biofilms. Once C4-HSL is sensed, the protein CheZ is produced, which causes the microbe to move in a straight-line path. As higher concentrations of C4-HSL are found, the microbe moves closer to the biofilm.

Destroy

After the engineered microbe is delivered to the biofilm, it must eliminate the fungal and bacterial contaminants present there. Cinnamaldehyde is used in order to permeate through the cell membrane since it is small enough to enter the phospholipid bilayer. However, fungi has additional protection cell wall that must be punctured to allow access to the membrane. The cell wall is anchored to the membrane by a polymer called chitin, so an additional enzyme called chitinase was used to open up the cell wall.

Solution

Once our three steps have been implemented, the engineered microbe will be dispersed through the biofilm. Theoretically, the cinnamaldehyde will kill all living microorganisms in the biofilm, including the engineered microbe. Thus, the biofilm will effectively be destroyed.

Result

With the sludge layer removed, biofuels will be a much more viable option for the consumer. With biofilms removed, fouling, degradation, and corrosion will be effectively eliminated from the storage process. This allows biofuels to be used more regularly resulting in a cheaper and healthier fuel for the world today.

Our Music Video!

We had a lot of fun working on our iGEM project, including the creation of our music video.